Uterine vascular remodeling occurs during gestation to meet increasing fetal nutrient demands. This remodeling includes modification of the uterine spiral arteries into low resistance vessels for supplying blood to the fetus. Central to uterine spiral artery remodeling are invasive trophoblast cells, known in the human as extravillous trophoblast (EVT). Impaired EVT development leads to suboptimal fetal conditions and adverse pregnancy outcomes including pregnancy loss, preeclampsia, intrauterine growth restriction, and preterm birth. We identified a critical and conserved regulator of EVT lineage development, Achaete-Scute Family Basic Helix-Loop-Helix Transcription Factor 2 (ASCL2). Depletion of ASCL2 in human trophoblast stem (hTS) cells inhibits EVT formation. Similarly, global depletion of ASCL2 in vivo disrupts placental development and causes embryonic lethality in the rat. However, the molecular mechanisms by which ASCL2 directs EVT lineage development are unknown. Our established hTS cell lines and protocols for generating mutant rat models will allow us to directly test our central hypothesis that ASCL2 controls EVT lineage development during placentation. To investigate higher order actions of ASCL2 on the epigenomic landscape of the EVT cell lineage, we will identify how ASCL2 depletion alters DNA methylation, chromatin accessibility and conformation using whole genome bisulfite sequencing (WGBS), assay for transposase-accessible chromatin-sequencing (ATAC-seq), and chromatin capture using Hi-C, respectively (Aim 1A). To identify direct genomic targets of ASCL2 we will perform chromatin immunoprecipitation sequencing (ChIP-seq) in EVT cells (Aim 1B). ASCL2 regulation of trophoblast development and invasion will then be evaluated in vivo using a newly established model of invasive trophoblast cell specific depletion based on breeding Prl7b1 Cre recombinase and Ascl2 floxed rats (Aim 2A). To examine ASCL2-positive trophoblast cell development in normal and diseased rat placentas we will conduct single cell RNA-sequencing (scRNA-seq) and single cell ATAC-seq (Aim 2B). The proposed research plan will provide the candidate with a body of experimental work necessary for independent publications and preliminary data for R-series grants. During the R00 phase the candidate will develop independence from her mentors by identifying targets of ASCL2 and investigating their contributions to invasive trophoblast lineage development with innovative rat models. The proposed research project serves as the foundation for the candidate’s long-term career goal of identifying how dysregulated spiral artery remodeling leads to­ a spectrum of diseases ranging from fetal growth restriction to preeclampsia. ­

K99HD107262

2022-08-02

2023-08-31